Prenatal Alcohol Exposure and Chronic Mild Stress Differentially Alter Depressive‐ and Anxiety‐Like Behaviors in Male and Female Offspring

Background: Fetal Alcohol Spectrum Disorder (FASD) is associated with numerous neurobehavioral alterations, as well as disabilities in a number of domains, including a high incidence of depression and anxiety disorders. Prenatal alcohol exposure (PAE) also alters hypothalamic‐pituitary‐adrenal (HPA) function, resulting in increased responsiveness to stressors and HPA dysregulation in adulthood. Interestingly, data suggest that pre‐existing HPA abnormalities may be a major contributory factor to some forms of depression, particularly when an individual is exposed to stressors later in life. We tested the hypothesis that exposure to stressors in adulthood may unmask an increased vulnerability to depressive‐ and anxiety‐like behaviors in PAE animals. Methods: Male and female offspring from prenatal alcohol (PAE), pair‐fed (PF), and ad libitum‐fed control (C) treatment groups were tested in adulthood. Animals were exposed to 10 consecutive days of chronic mild stress (CMS), and assessed in a battery of well‐validated tasks sensitive to differences in depressive‐ and/or anxiety‐like behaviors. Results: We report here that the combination of PAE and CMS in adulthood increases depressive‐ and anxiety‐like behaviors in a sexually dimorphic manner. PAE males showed impaired hedonic responsivity (sucrose contrast test), locomotor hyperactivity (open field), and alterations in affiliative and nonaffiliative social behaviors (social interaction test) compared to control males. By contrast, PAE and, to a lesser extent, PF, females showed greater levels of “behavioral despair” in the forced swim test, and PAE females showed altered behavior in the final 5 minutes of the social interaction test compared to control females. Conclusions: These data support the possibility that stress may be a mediating or contributing factor in the psychopathologies reported in FASD populations.     

Enzastaurin (LY317615), a protein kinase Cbeta inhibitor, inhibits the AKT pathway and induces apoptosis in multiple myeloma cell lines

Enzastaurin (LY317615), an acyclic bisindolylmaleimide, is an oral inhibitor of the protein kinase Cbeta isozyme. The objective of this study was to assess the efficacy of enzastaurin in inducing apoptosis in multiple myeloma (MM) cell lines and to investigate possible mechanisms of apoptosis. Cell proliferation assays were done on a variety of MM cell lines with unique characteristics (dexamethasone sensitive, dexamethasone resistant, chemotherapy sensitive, and melphalan resistant). The dexamethasone-sensitive MM.1S cell line was used to further assess the effect of enzastaurin in the presence of dexamethasone, insulin-like growth factor-I (IGF-I), interleukin-6, and the pan-specific caspase inhibitor ZVAD-fmk. Enzastaurin increased cell death in all cell lines at clinically significant low micromolar concentrations (1-3 micromol/L) after 72 hours of treatment. Dexamethasone and enzastaurin were shown to have an additive effect on MM.1S cell death. Although IGF-I blocked the effect of 1 micromol/L enzastaurin, IGF-I did not abrogate cell death induced with 3 mumol/L enzastaurin. Moreover, enzastaurin-induced cell death was not affected by interleukin-6 or ZVAD-fmk. GSK3beta phosphorylation, a reliable pharmacodynamic marker for enzastaurin activity, and AKT phosphorylation were both decreased with enzastaurin treatment. These data indicate that enzastaurin induces apoptosis in MM cell lines in a caspase-independent manner and that enzastaurin exerts its antimyeloma effect by inhibiting signaling through the AKT pathway.     

Fibrinopeptide A and the phosphate content of fibrinogen in venous thromboembolism and disseminated intravascular coagulation

Concentrations of plasma fibrinopeptide A (FPA) were measured by radioimmunoassay in 50 patients with venous thromboembolism or disseminated intravascular coagulation or both. A consistent discrepancy was observed in values obtained with two anti-FPA antisera. Analysis of extracts from plasma of these patients by high-performance liquid chromatography (HPLC) revealed the presence of a phosphorylated and an unphosphorylated form of the A peptide. Differences in concentrations of FPA measured with the two antisera could be accounted for by their different reactivity with phosphorylated FPA (FPA-P). The differences were abolished by treatment with alkaline phosphatase. A good correlation was observed between the FPA-P content of free A- peptide material and of fibrinogen in plasma as determined by HPLC (r = .88, P less than .001, n = 11). In patients with elevated FPA levels, the mean FPA-P content of fibrinogen was significantly higher (P less than .002, n = 13) than in patients with normal FPA levels (n = 8) and in healthy controls (n = 14). Phosphorus in fibrinogen did not correlate with fibrinogen degradation products or fibrinogen levels and became normal on adequate anticoagulation. Therefore, blood-clotting activation may lead to a high phosphate content of fibrinogen and of free FPA in plasma.     

Autoantibodies to the high-affinity IgE receptor in children with chronic urticaria.

BACKGROUND: Chronic urticaria (CU) in childhood remains a challenge for investigation, and its etiology is largely unknown. Autoantibodies to the high-affinity IgE receptor (FcepsilonRI) are believed to play a role in the pathogenesis of this disease in adults. OBJECTIVE: To determine the prevalence of autoantibodies to FcepsilonRIalpha on basophils in children with CU vs atopic eczema dermatitis syndrome (AEDS). METHODS: Eighty children with CU were compared with 38 children with AEDS. In addition to complete blood cell counts and total IgE measurements, CAP-RASTs to egg, codfish, soy, milk, and peanut were performed. Stool samples were examined for parasites, and autologous serum skin testing and a functional anti-FcepsilonRIalpha assay were conducted to detect autoantibodies. RESULTS: No significant differences were observed between children with CU and controls in mean basophil or eosinophil counts. Twenty (26%) of 77 children with CU and 31 (82%) of 38 with AEDS had positive CAP-RAST results (P < .001). Only 2.5% of the children with CU and 0% with AEDS had stool samples positive for parasites (P = .005). Anti-FcepsilonRIalpha autoantibodies were positive in 37 (47%) of 78 children with CU and in none of 33 with AEDS. Non-IgG histamine-releasing factors were found in 10 (13%) of 78 children with CU. CONCLUSIONS: Children have a similar prevalence of autoantibodies to the FcepsilonRIalpha as has been previously published for adults. Few have type I allergies, and parasite infestation is also uncommon. Further studies are required to investigate the predictive value of the autoantibodies in these children with respect to clinical profile, requirements for medications other than antihistamines, and remission rates.     

Optimization and Limitations of Use of Cryopreserved Peripheral Blood Mononuclear Cells for Functional and Phenotypic T-Cell Characterization

The goals of this study were to optimize processing methods of cryopreserved peripheral blood mononuclear cells (PBMC) for immunological assays, identify acceptance parameters for the use of cryopreserved PBMC for functional and phenotypic assays, and to define limitations of the information obtainable with cryopreserved PBMC. Blood samples from 104 volunteers (49 human immunodeficiency virus-infected and 55 uninfected) were used to assess lymphocyte proliferation in response to tetanus, candida, and pokeweed-mitogen stimulation and to enumerate CD4⁺ and CD8⁺ T cells and T-cell subpopulations by flow cytometry. We determined that slowly diluting the thawed PBMC significantly improved viable cell recovery, whereas the use of benzonase improved cell recovery only sometimes. Cell storage in liquid nitrogen for up to 15 months did not affect cell viability, recovery, or the results of lymphocyte proliferation assays (LPA) and flow cytometry assays. Storage at −70°C for ≤3 weeks versus storage in liquid nitrogen before shipment on dry ice did not affect cell viability, recovery, or flow cytometric results. Storage at −70°C was associated with slightly higher LPA results with pokeweed-mitogen but not with microbial antigens. Cell viability of 75% was the acceptance parameter for LPA. No other acceptance parameters were found for LPA or flow cytometry assay results for cryopreserved PBMC. Under optimized conditions, LPA and flow cytometry assay results for cryopreserved and fresh PBMC were highly correlated, with the exception of phenotypic assays that used CD45RO or CD62L markers, which seemed labile to freezing and thawing.Utilization of cryopreserved peripheral blood mononuclear cells (PBMC) for immunologic assays has dramatically increased in recent years. Cryopreserved PBMC are particularly useful in clinical trials with low end-point frequencies because they allow the immunologic assays to be performed after the conclusion of the studies, when all the end points have already been identified (1, 14, 18). In addition, the use of cryopreserved PBMC allows all assays to be performed in a single laboratory, eliminating interlaboratory variability, which has been a confounder in some studies (15). Furthermore, if changes in immunologic parameters over time are the main outcome of the immunologic studies, interassay variability may become a confounder, too. Testing all the cryopreserved PBMC per subject at one time can eliminate this confounder.The use of cryopreserved PBMC in immunological assays poses challenges, including the availability of adequate equipment (7) and the need for technical proficiency. Assays have to be adapted and validated for the use of cryopreserved PBMC (4, 6, 9, 11, 17, 19), and the quality of the frozen cells has to be monitored (5) to ensure reliable results in functional and phenotypic assays.The Cryopreservation Working Group (WG) of the Pediatric AIDS Clinical Trials Group (PACTG), which operated between 1999 and 2006, developed a series of experiments aimed at optimizing methods of PBMC cryopreservation for assays requiring viable cells, adapting immunologic assays to cryopreserved PBMC, and establishing quality control parameters for immunologic assays with cryopreserved PBMC. The group focused on highly complex functional and phenotypic assays commonly used as outcome measures in studies that address immune suppression or reconstitution. Unlike previous studies of cryopreserved PBMC in immunologic assays, which were done at single laboratories (3, 8, 13, 17), our study presents results obtained at eight laboratories, substantiating the generalizibility of our findings.     

Successful immune reconstitution decreases leukemic relapse and improves survival in recipients of unrelated cord blood transplantation.

Allogeneic hematopoietic stem cell transplantation (HSCT) is established therapy for selected patients with acute leukemia. After transplantation, antileukemic immune responses are believed to eliminate residual leukemia cells and decrease the likelihood of relapse. However, the clinical effect of successful antigen-specific immune reconstitution after HSCT on the likelihood of leukemic relapse and overall survival is not known. Pediatric recipients of unrelated cord blood transplants who underwent transplantation for acute leukemia were sequentially evaluated for their development of antigen-specific T-lymphocyte immunity to herpes viruses. The clinical effect of a positive antigen-specific response on relapse-free survival was determined. The presence of an antigen-specific response resulted in a relapse-free survival advantage (P = .0001), which was primarily due to a decrease in leukemic relapse (P = .003). Proportional hazards modeling for time to relapse and time to relapse or death defined 3 variables that were strongly associated with a poor outcome: female gender, poor remission status before transplantation, and negative antigen-specific T-lymphocyte proliferation. Notably neither acute nor chronic graft-versus-host disease had any effect on the incidence of leukemic relapse. Successful antigen-specific immune reconstitution after unrelated cord blood transplantation results in decreased leukemic relapse and improved overall survival.